High content cellular immune profiling reveals differences between rhesus monkeys and men

Abstract

A better understanding of similarities and differences in the composition of the cellular immune system in non-human primates (NHPs) compared with human subjects will improve the interpretation of preclinical studies. It will also aid in addressing the usefulness of NHPs as subjects for studying chronic diseases, vaccine development and immune reconstitution. We employed high content colour flow cytometry and analysed simultaneously the expression of CD3, CD4, CD8alpha, CD8beta, CD16/CD56, CD45RA, CCR7, CD27, CD28, CD107a and the interleukin-7 receptor alpha-chain (IL-7Ralpha) in peripheral blood mononuclear cells (PBMCs) of 27 rhesus macaques and 16 healthy human subjects. Regulatory T cells (Tregs) were identified using anti-CD3, -CD4, -CD25, -FoxP3, and -IL-7Ralpha monoclonal antibodies. Responsiveness to IL-7 was gauged in a signal transducer and activation of transcription 5 (STAT-5) phosphorylation assay. Human and NHP PBMCs showed a similar T-cell composition pattern with some remarkable differences. Similarities: human and NHP CD4(+) and CD8(+) cells showed a similar STAT-5 phosphorylation pattern in response to IL-7. Multicolour flow cytometric analysis identified a CD4(+) CD8alphaalpha(+) CD8alphabeta(+) T-cell population in NHPs as well as in human subjects that expressed the degranulation marker CD107a and may represent a unique CD4(+) T-cell subset endowed with cytotoxic capacity. Differences: we identified in PBMCs from NHPs a higher proportion (5.16% in CD3(+) T cells) of CD8alphaalpha(+) T cells when compared with human donors (1.22% in CD3(+) T cells). NHP CD8alphaalpha(+) T cells produced tumour necrosis factor-alpha / interferon-gamma (TNF-alpha/IFN-gamma) or TNF-alpha, whereas human CD8alphaalpha(+) T cells produced simultaneously TNF-alpha/IFN-gamma and IL-2. A minor percentage of human CD8(+) T cells expressed CD25(bright) and FoxP3 (0.01%). In contrast, 0.07% of NHP CD8(+) T cells exhibited the CD25(bright) FoxP3(+) phenotype. PBMCs from NHPs showed less IL-7Ralpha-positive events in all T-cell subsets including CD4(+) Tregs (median 5%) as compared with human (median 12%). The data visualize commonalities and differences in immune cell subsets in humans and NHPs, most of them in long-lived memory cells and cells with suppressive functions. This provides a matrix to assess future efforts to study diseases and vaccines in NHPs.

Figure 1

Identification of T-cell subsets. (a) Gating strategy to identify T cells, based on expression of CD3, CD8α, CD8β and CD4. (b) T-cell frequencies. CD8αα⁺, CD4⁺ CD8αα⁺ and CD4⁺ CD8αβ⁺ T-cell frequencies were statistically higher in non-human primates. P <0·001 (Mann–Whitney U-test).

Figure 2

Overview of T-cell subsets defined by CD45RA/CCR7 and CD27/CD28 expression using heat-map analysis. (a) Frequency of immune cell subsets in human donors and non-human primates. (b) Interleukin-7 receptor α (IL-7Rα) expression and relative IL-7Rα density [as measured by mean fluorescence intensity (MFI)] in these T-cell subsets. The percentage of IL-7Rα (and MFI) in T-cell subsets displaying low number of events (< 100 events) was not determined (n.d.) for quality control reasons.

Figure 3

Cytokine producing T-cell subsets in human donors and non-human primates. (a) Presence of immune cell subsets defined by CD3, CD4, CD8α and CD8β expression with no stimulation (medium control) and phorbol 12-myristate 13-acetate (PMA)/ionomycin stimulation. (b) Percentage of cytokine producing T cells. **P <0·001, *P <0·05 (Mann–Whitney U-test).

Figure 4

Analysis of polyfunctional T cells. Interleukin-2 (IL-2), interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) production were measured by intracellular cytokine staining on the single-cell level. Two representative individuals from human donors and non-human primates are shown as a paradigm. Note the increased frequency of polyfunctional T-cells in human donors compared with non-human primates.

Figure 5

Interleukin-7 (IL-7) -induced signal transducer and activator of transcription 5 (STAT-5) phosphorylation. Phosphorylated (P-) STAT-5 was determined in CD4⁺ and CD8⁺ cells before and after exposure to IL-7. (a) Percentage of P-STAT-5 positive cells. Similar levels of constitutive and IL-7-induced STAT-5 phosphorylation in peripheral blood mononuclear cells from human donors and non-human primates. (b) Example of constitutive and IL-7-induced P-STAT-5 in human and non-human primate peripheral blood mononuclear cells determined by flow cytometry.

Figure 6

Determination of regulatory T cells based on marker analysis. (a) CD4⁺ CD8^– (left), CD4⁺ CD8⁺ (middle panel) and CD8⁺ CD4⁻ (right) T cells were segregated based on CD25 expression and FoxP3 analysis was performed as shown in the supplementary Fig. S2. Note that some CD8⁺ (CD4⁻) CD25^bright T-cells showed low FoxP3 expression in non-human primates. (b) Interleukin-7 receptor α (IL-7Rα) -expressing T cells in CD4⁺ CD8⁻ regulatory T cells (Tregs) based on FoxP3 analysis. Higher percentage of IL-7Rα⁺ T cells in Foxp3⁻ CD25^bright Tregs in peripheral blood mononuclear cells from humans than in those from non-human primates. **P <0·001, *P <0·05 (Mann–Whitney U-test).

Figure 7

Model of CD3⁺ CD4⁺ CD8⁺ T-cell differentiation. Human donor and non-human primate CD3⁺ CD4⁺ CD8αα⁺ and CD3⁺ CD4⁺ CD8αβ⁺ T cells expressed CD107a and displayed a very similar phenotype to CD3⁺ CD4⁺, suggesting that CD3⁺ CD4⁺ CD8⁺ arise from CD3⁺ CD4⁺ and represent a ‘back-up’ compartment endowed with cytotoxic functions. CD4⁺ CD8αα⁺ may arise from CD4⁺ CD8αβ⁺ (or vice versa).